1. Field of the Invention
The present invention concerns a method of sensitizing multidrug resistant (MDR) cells to antitumor agents using phenothiazines (PTZs) and thioxanthenes.
2. Background of the Invention
Phenothiazines and structurally related antipsychotic agents inhibit several cellular enzymes and block the function of critical cellular receptors (Roufogalis, B. D. "Specificity of Trifluoperazine and Related Phenothiazines for Calcium Binding Proteins", In: W. Y. Cheung (ed.) Calcium and Cell Function, Vol III, pp. 129-159, New York, Academic Press, 1982; Pang D. C. and Briggs, F. N., "Mechanism of Quinidine and Chlorpromazine Inhibition of Sarcotubular ATPase Activity, Biochem. Pharmacol., 25, 21-25, (1976); Ruben, L. and Rasmussen, H., "Phenothiazines and Related Compounds Disrupt Mitochondrial Energy Production by a Calmodulin-Independent Reaction", Biochim. Biophys.Acta., 637, 415-422 (1981); Creese, I., and Sibley, D. R., "Receptor Adaptations to Centrally Acting Drugs", Ann.Rev. Pharmacol. Toxicol., 21, 357-391, (1980)).
Prominant among the cellular targets is calmodulin (CaM), the multifunctional calcium binding protein (Levin, R. M., and Weiss, B., "Mechanism By Which Psychotropic Drugs Inhibit Adensine Cyclic 3',5'-monophosphate PDE of Brain", Mol. Pharmacol., 12, 581-589 (1976)).
CaM has been implicated in the regulation of numerous cellular events (Manalan, A. S. and C. B. Klee, "Calmodulin", Advances In Cyclic Nucleotide Protein Phosphorylation Res., 18,227-278 (1984) including that of normal (Veigl, M. L. Vanaman, T. C., and Sedwick, W. D., "Calcium and Calmodulin in Cell Growth and Transformation", Biochem. Biophys. Acta., 738, 21-48, (1984)) and abnormal cellular proliferation (Hait, W. N. and Lazo, J. S. "Calmodulin, "A Potential Target for Cancer Chemotherapeutic Agents", J. Clin. Oncol., 4, 994-1012 (1986)); Rasmussen, C. D. and Means, A. R., "Calmodulin - Regulation of Cell Proliferation", EMBO, 6, 3961-3968 (1987); Wei, J. W., R. A. Hickie, and D. J. Klaassen, "Inhibition of Human Breast Cancer Colony Formation by Anticalmodulin Agents: Trifluoperazine, W-7, and W-13", Cancer Chemother. Pharmacol., 11, 86-90 (1983)). Consistent with these observations was the demonstration that PTZs and other CaM antagonists possess antiproliferative and cytotoxic effects (Ito, H., and H. Hidaka, "Antitumor Effect of a Calmodulin Antagonist on the Growth of Solid Sarcoma", 180, Cancer Lett. 19, 215-220 (1983)) that were proportional to their anti-CaM activity (Hait, W. N., Grais, L., Benz, C., Cadman, E., "Inhibition of Growth of Leukemic Cells by Inhibitors of Calmodulin: Phenothiazines and Melittin", Cancer Chemother. Pharmocol., 14, 202-205 (1985)).
The recent demonstration and elucidation of the phenomenon of multidrug resistance (MDR) has led to the search for drugs that could sensitize highly resistant cancer cells to chemotherapeutic agents. MDR is the process whereby malignant cells become resistant to structurally diverse chemotherapeutic agents following exposure to a single drug (Riordan, J. R., and V. Ling, "Genetic and Biochemical Characterization of Multidrug Resistance", Pharmol. Ther., 28., 51-75 (1985)). MDR cell lines classically have been associated with decreased drug accumulation due to enhanced efflux as well as diminished influx of chemotherapeutic drugs (Inaba M., and R. K. Johnson, "Uptake and Retention of Adriamycin and Daunorubicin by Sensitive and Anthracycline-Resistant Sublines of P388 Leukemia", Biochem. Pharmacol., 27, 2123-2130 (1978) and Fojo, A. S. Akiyama, M. M. Gottesman, and I. Pastan, "Reduced Drug Accumulation in Multiple-Drug Resistant Human KB Carcinoma Cell Lines", Cancer Res., 45, 3002-3007 (1985)). This effect appears to be attributable to the overexpression of a 170,000 dalton membrane glycoprotein (P-glycoprotein) which structurally resembles transport proteins in prokaryotic cells (Chen C., J. E. Chin, K. Ueda, C. P. Clark, I. Pastan, M. M. Gottesman, and I. B. Roninson, "Internal Duplication and Homology with Bacterial Transport Proteins in the mdrl (P-glycoprotein) Gene from Multidrug Resistant Human Cells", Cell, 47, 381-389 (1986)) and may function as an energy-dependent, drug efflux pump in mammalian cells (Hamada, H., and T. Tsuruo, "Purification of the 170- to 180-Kilodalton Membrane Glycoprotein Associated with MDR; 170-to 180-Kilodalton Membrane Glycoprotein is an ATPase, J. Biol. Chem, 263, 1454-1458 (1988) and Akiyama, S., M. M. Cornwell, M. Kuwano, I. Pastan, and M. M. Gottesman, "Most Drugs that Reverse Multidrug Resistance Inhibit Also Photoaffinity Labeling of P-glycoprotein by a Vinblastine Analog", Mol. Pharmacol., 33, 144-147 (1988)).
PTZs have been shown to be among the group of drugs known to modify MDR (Ganapathi, R., and D. Grabowski, "Enhancement of Sensitivity to Adriamycin in Resistant P388 Leukemia by the Calmodulin Inhibitor Trifluoperazine", Cancer Res., 43, 3696-3699 (1983) and Akiyama, S. N. Shiraishi, Y. Kuratomi, M. Nakagowa, and M. Kuwano, "Circumvention of Multiple-Drug Resistance in Human Cancer Cells by Thioridazine, Trifluoperazine and Chlorpromazine", J. Natl. Cancer Inst., 76: 839-844 (1986)). Although the mechanism by which PTZs and other drugs modulate MDR is not yet clear, it has been suggested that their pharmacological properties may be mediated by the calcium messenger system, since the active compounds are known to inhibit voltage-dependent calcium channels (Fleckenstein, A., "Specific Pharmacology of Calcium in Myocardium, Cardiac Pacemakers, and Vascular Smooth Muscle", Ann. Rev. Pharmacol. Toxicol., 17, 149-166 (1977), CaM and protein kinase C (Mori, T., Y. Takai, R. Minakuchi, B. Yu, and Y. Nishizuka, "Inhibitory Action of Chlorpromazine, Dibucaine, and other Phospholipid-Interacting Drugs on Calcium-activated, Phospholipid-Dependent Protein Kinase", J. Biol. Chem., 255 8378-8380 (1980).
Prozialeck, W. C. and Weiss, B., "Inhibition of Calmodulin by Phenothiazines and Related Drugs: Structure-Activity Relationships", J. Parmacol. Exp. Ther., 222, 509-516 (1982) studied the specific structural features which influence the interaction of a large number of PTZ derivatives with CaM, and showed that varying either the PTZ nucleus or the amino side chain altered activity. Specifically, ring-substitutions that increased hydrophobicity increased potency, while modifications of the type or length of the amino side chain affected potency in a manner unrelated to hydrophobicity. Similarly, studies with N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (Hidaka, H., M. Asano, and T. Tanaka, "Activity-structure Relationship of Calmodulin Antagonists", Mol. Pharmacol, 20, 571-578 (1981)) and a series of 15 derivatives of W-7 (MacNeil, S., M. Griffin., A. M. Cooke, N. J. Pettett, R. A. Dawson, R. Owen, and G. M. Blackburn, "Calmodulin Antagonists of Improved Potency and Specificity for Use in the Study of Calmodulin Biochemistry", Biochem. Pharmacol., 37, 1717-1723 (1988)) demonstrated that both halogenation of the naphthalene ring with chlorine, iodine or cyano groups, and increasing the length of the alkyl side chain from 4 to 12 carbons increased their potency against CaM.
Drug binding studies with synthetic peptides and molecular modeling provided a rationale for the importance of both hydrophobicity and molecular structure for the PTZ-CaM interaction. The induction of alpha-helix formation by the binding of Ca.sup.2+ to CaM results in two distinct regions, a hydrophobic pocket containing two aromatic phenylalanine residues (Phe 89 and 92) oriented to form a charge transfer complex with the aromatic, tricyclic nucleus of the PTZs, and a hydrophilic region at a distance of one-half helical turn formed by glutamic acid residues (Glu 83,84 and 87), which interact with the positively-charged nitrogen atom of the PTZ side chain (Reid, R. E., "Drug Interactions with Calmodulin: The Binding Site", J. Thero. Biol., 105, 63-76 (1983)).
In Johnston et al, The Lancet, Apr. 22, 1978 "Mechanism of the Anti-psychotic Effect in the Treatment of Acute Schizophrenia" pp. 842-851 (1978), a clinical trial of the antipsychotic effects of cis-flupenthixol versus trans-flupenthixol versus placebo showed that while cis-flupenthoxil was a potent neuroleptic (especially for "positive" symptoms), trans-flupenthixol had no activity as an anti-psychotic. Since trans-fluopenthixol is a far less potent dopamine antagonist, and the extrapyramidal side effects associated with antipsychotic therapy are attributed to dopamine receptor binding, trans-flupenthixol lacks these side effects. The apparent lack of anti-psychotic activity or extrapyramidal side effects of trans-flupenthixol make it particularly attractive for use as an anti-multidrug resistance agent, since it is these side effects which have proven problematic in reported trials of phenothiazines plus doxorubicin.
Flupenthixol is disclosed in U.K. Patent 925,538 as having utility as a tranquilizer, ataractic, antiemetic, antihistamine, antispasmodic and general central nervous system depressant. No mention is made of any antitumor activity.
Several thioxanthene derivatives are disclosed in U.K. Patent 863,699 as tranquilizers. Again, no mention of anti-tumor activity is made.
Robin L. Miller, et al, Journal of Clinical Oncology, Vol 6, No. 5, May 1988, 880-888 demonstrated the possible effectiveness of trifluperazine (a phenothiazine) in combination with doxorubicin in a Phase I/II trial in clinically resistant cancer in humans. The dose limiting factor in these trials was the extrapyramidal side effects associated with trifluperazine.